Phospholipid ether analogs as cancer-targeting drug vehicles

ABSTRACT

The present invention is directed to therapeutic compounds capable of targeting cancer cells and cancer stem cells. The present invention is further directed to compositions comprising these therapeutic compounds and methods of treating cancer comprising administering these therapeutic compounds.

CROSS-REFERENCE TO REALTED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.14/934,209, filed Nov. 6, 2015 (now U.S. Pat. No. 9,345,718, issued May24, 2016), which claims the benefit of Provisional Application No.62/080,436, the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

In 2012, 14.1 million people were diagnosed with cancer worldwide and8.2 million died of cancer. In the United States, around 40% of allpeople will be diagnosed with cancer during their lifetime. Despitereceiving the best treatment available, 44% of those Americans will diefrom cancer.

Cancer is the result of a cell dividing without limitation. Healthycells have checkpoints that prevent unlimited cell division. A fewexamples of these checkpoints are nutrient availability, DNA damage andcontact inhibition (i.e. a cell comes into contact with another cell).Additionally, most cells can replicate only a finite number of times andthus are programmed to die after a particular number of cell divisions.

Cancer is the result of a cell overcoming these built-in checkpoints andproliferating beyond control. This uncontrolled proliferation leads tothe formation of a tumor. There are two types of tumors, benign andmalignant. Benign tumors are incapable of crossing natural boundariesbetween tissue types. Malignant tumors, on the other hand, are capableof invading nearby tissue or entering the bloodstream and metastasizingto a different location. Only malignant tumors are considered cancerous.It is this ability to infiltrate and metastasize that makes cancer sucha deadly disease.

To further complicate the fight against cancer, malignant tumors havedistinct cell types. One particularly troublesome type is cancer stemcells (“CSC's”). CSC's are capable of self-renewing and differentiatinginto the distinct types of cancer cells found in a malignant tumor.Thus, CSC's are a primary factor in the metastatic ability of a tumor.CSC's often survive radiation and chemotherapy. It is hypothesized thatrecurrence of cancer after radiation and chemotherapy is the result ofthe inability of radiation and chemotherapy to kill all CSC's combinedwith the ability of CSC's to establish a new tumor.

A particularly troublesome type of cancer is brain cancer. Braincancers, such as high-grade gliomas, are often treated with surgeryfollowed by radiation therapy. Surgery for brain tumors is often verycomplicated. The surgeon must remove the tumor without damaging anynearby brain tissue that could result in physical or cognitivedisabilities. Often the surgeon is incapable of removing the boundariesof the tumor that contact the healthy tissue. Radiation therapy is oftenused to kill these remaining cancer cells. However, radiation doses arelimited by the potential damage to healthy brain tissue. Unfortunately,brain cancer is usually chemotherapy resistant. This resistance islargely attributable to the blood-brain barrier (“BBB”). The BBB is aphysical barrier that separates the fluid surrounding the brain fromblood cells and other components in the blood stream. Most anti-cancerdrugs are unable to cross the BBB.

One method of treating brain cancer is to inhibit the growth of newblood vessels that are necessary for tumor size progression. Bevacizumabmarketed under the trademark Avastin® (Avastin is a registered trademarkof Genentech, Inc.) is used to stop and even reverse tumorvascularization. However, Rich J., and colleagues, Canc Res, 2006, 66,7843, found that when Avastin® was used to treat a glioma stem cellderived brain tumor it resulted in hypoxia and a lowered pH.Sathornsumetee S., Phase II trial of bevacizumab and erlotinib inpatients with recurrent malignant glioma, Neuro-Oncol, 2010, December,12(12), 1300-1310. Hypoxia and low pH are both known to cause CSCpropagation and can promote CSC-driven tumor recurrence.

Chemotherapy is a term used to describe a particular type of cancertreatment that includes using cytotoxic anti-cancer drugs. Cytotoxicdrugs used during chemotherapy can be broken down into several maincategories including alkylating agents, antimetabolites, anti-tumorantibiotics, topoisomerase inhibitors, and mitotic inhibitors. Cytotoxicanti-cancer drugs typically cause cell division to cease and thus affecthealthy tissue as well as cancerous tissue. Alkylating agents stopcancer cell division by damaging the DNA of the cancer cell. Some commonalkylating agents used to treat cancer are nitrogen mustards (e.g.cyclophosphamide (Cytoxan®; Cytoxan is a registered trademark of BaxterInternational), nitrosoureas, alkyl sulfonates, triazeines, andethylenimines. Platinum drugs, such as cisplatin and carboplatin, worksimilarly to alkylating agents. Antimetabolites stop cancer celldivision by inhibiting DNA and RNA synthesis. Some commonantimetabolites used to treat cancer are 6-mercaptopurine, gemcitabine(Gemzar®; Gemzar is a registered trademark of Eli Lilly and Company),methotrexate and pemetrexed (Alimta®; Alimta is a registered trademarkof Eli Lilly and Company). Topoisomerase inhibitors stop cancer celldivision by inhibiting topoisomerase enzymes from separating the DNA forreplication. Some common topoisomerase inhibitors are topotecan,irinotecan, etoposide, and teniposide. Mitotic inhibitors stop cancercell division by inhibiting key cell division enzymes. Some commonmitotic inhibitors are taxanes (e.g. paclitaxel (Taxol®; Taxol is aregistered trademark of Bristol-Myers Squibb Company) and docetaxel(Taxotere®; Taxotere is a registered trademark of Aventis Pharma SA)),epothilones, and vinca alkaloids.

One disadvantage of all of these anti-cancer drugs is the damage thatthey do to healthy tissue. Because the drugs treat cancer by inhibitingnormal cell function, healthy tissue that also relies on constant celldivision such as blood cells, mucosal surfaces and skin, can be severelydamaged as well. This damage results in significant morbidity and canlimit the amount of chemotherapy that can safely be delivered. Examplesof side effects that occur during chemotherapy treatment include lowblood count, hair loss, muscle and joint pain, nausea, vomiting,diarrhea, mouth sores, fever, and chills. To overcome this problem drugsare being developed that affect proteins and cellular functions thatoccur only in cancer cells. Some of these specific cancer drugs areimatinib (Gleevec®; Gleevec is a registered trademark of Novartis AG),gefitinib (Iressa®, Iressa is a registered trademark of AstraZeneca UKLimited), sunitinib (Sutent®; Sutent is a registered trademark of C.P.Pharmaceuticals, International C.V.), and bortezomib (Velcade®; Velcadeis a registered trademark of Millennium Pharmaceuticals, Inc.). However,these drugs are not approved for the treatment of all cancer types andare universally associated with the development of treatment resistance.Thus, a need exists in the art for an anti-cancer drug delivery vehiclethat can deliver potent, effective, broad spectrum anti-cancer drugs tocancer cells including CSC's while avoiding substantial uptake of thedrug by healthy cells. Additionally, the anti-cancer drug deliveryvehicle should be able to cross the BBB and deliver the anti-cancer drugto cancer cells of the brain.

Currently, there are few chemical compounds that preferentially targetcancer cells. One such compound is CLR1404. Generally, CLR1404 is apromising new tumor-selective diagnostic imaging agent used to monitorthe treatment response of several tumor treatment modalities.Radioiodinated CLR1404, a second-generation phospholipid ether (“PLE”)analog

with the following structure,

has displayed remarkable tumor selectivity in 55/60 xenograft,orthotopic and transgenic cancer and cancer stem cell derived animalmodels making the core molecule an ideal platform for an anti-cancerdrug delivery vehicle. See U.S. Pat. No. 8,535,641; U.S. PatentApplication Publication No. 2014/0030187 and Weichert, J. P., et al.,Alkylphosphocholine analogs for broad-spectrum cancer imaging andtherapy, Sci Transl Med, 2014, Jun. 11, 6(240), 240ra75; each of whichare incorporated by reference herein in its entirety.

What is not known is whether a compound that is selectively sequesteredand retained by cancer cells and cancer stem cells is capable ofdelivering an anti-cancer drug to these same cells. Further, it is notknown whether this compound is also capable of transporting anti-cancerdrugs across the BBB to treat brain cancers. Finally, it is unknownwhether this or similar compounds can cause the cancer cell to retainthe anti-cancer drug in sufficient quantities and for a sufficientperiod of time to eradicate the tumor and prevent further growth andmetastasis. The present invention adapts the CLR1404 core molecule foruse as an anti-cancer drug delivery vehicle capable of targeting theanti-cancer drug to cancer cells and cancer stem cells including braincancer cells. Further, the compounds of the present invention areretained in cancer cells.

SUMMARY OF THE INVENTION

The present invention is directed to therapeutic compounds capable oftargeting cancer cells and cancer stem cells including brain tumorcells. The present invention is also directed to therapeutic compoundscapable of being sequestered and retained by cancer cells and cancerstem cells including brain tumor cells in sufficient quantity and forsufficient duration to treat the cancer and prevent metastasis andrecurrence.

In one embodiment, the present invention is directed to a therapeuticcompound of the formula A-B-D wherein:

-   -   A is at least one compound of formula (I),

-   -   at least one compound of formula (II),

-   -   at least one compound of formula (III),

-   -   or a combination thereof,    -   wherein W is selected from the group consisting of an aryl, a        C₁-C₆ alkyl, an alkenyl, an optionally substituted C₃-C₆        cycloalkyl and an optionally substituted C₃-C₆ heterocycloalkyl,        wherein R is H or an alkyl and wherein m is an integer from 12        to 24;    -   B is a linker compound, preferably a bond or a compound of        formula (IV), Y—(CH₂)_(n)—Z (IV), wherein:        -   Y is bound to A;        -   Z is bound to D;        -   Y is selected from the group consisting of a bond, O, NH,            C═O, NHSO₂O, and OC(═O)O;        -   Z is selected from the group consisting of O, NH, C═O,            C(═O)O, C(═O)NH, SO₂, OC(═O)OCH₂, and —S—S—; and        -   n is an integer from 0 to 6; and    -   D is an anti-cancer drug,        wherein the ratio of A to D is from 1:2 to 2:1.

In another embodiment, the present invention is directed to atherapeutic compound of the formula A-B-D selected from the groupconsisting of

and a combination thereof, wherein:

-   -   W is selected from the group consisting of an aryl, a C₁-C₆        alkyl, an alkenyl, an optionally substituted C₃-C₆ cycloalkyl        and an optionally substituted C₃-C₆ heterocycloalkyl;    -   R is H or an alkyl;    -   m is an integer from 12 to 24;    -   Y is selected from the group consisting of a bond, O, NH, C═O,        NHSO₂O, and OC(═O)O;    -   Z is selected from the group consisting of O, NH, C═O, C(═O)O,        C(═O)NH, SO₂, OC(═O)OCH₂, and —S—S—;    -   n is an integer from 0 to 6; and    -   D is an anti-cancer drug,        wherein B is optionally a bond between A and D.

In a preferred embodiment, the present invention is directed to atherapeutic compound of the formula A-B-D wherein:

-   -   A is compound of formula (I), wherein W is selected from the        group consisting of a C₁ alkyl,

-   -    and wherein m is 18;    -   B is a linker compound selected from a bond and a compound of        formula (IV), Y—(CH₂)_(n)—Z (IV), wherein n is an integer from 0        to 6, Y is bound to A, Z is bound to D, Y is selected from the        group consisting of a bond and C═O and Z is selected from the        group consisting of NH, C═O, C(═O)NH and C(═O)O; and    -   D is selected from the group consisting of paclitaxel,        irinotecan, topotecan, gemcitabine, cisplatin, geldanamycin and        mertansine.

In a more preferred embodiment, the present invention is directed to atherapeutic compound of the formula A-B-D wherein:

-   -   A is a compound of formula (I), wherein W is

-   -    and m is 18;    -   B is a compound of formula (IV), wherein Y is C═O and Z is C═O,        C(═O)NH or C(═O)O    -   and n is 3 or 4; and    -   D is paclitaxel,        wherein the ratio of A to D is 1:1.

In another more preferred embodiment, the present invention is directedto a therapeutic compound of the formula A-B-D wherein:

-   -   A is a compound of formula (I), wherein W is

-   -    and m is 18;    -   B is a bond or a compound of formula (IV), wherein Y is C═O, Z        is NH and n is 1 or 3;    -   and    -   D is geldanamycin,        wherein the ratio of A to D is 1:1.

In another more preferred embodiment, the present invention is directedto a therapeutic compound of the formula A-B-D wherein:

-   -   A is a compound of formula (I), wherein W is

-   -    and m is 18;    -   B is a bond; and    -   D is mertansine,        wherein the ratio of A to D is 1:1.

In another aspect, the present invention provides a pharmaceuticalcomposition containing a compound of the present invention incombination with one or more pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method of treatingcancer comprising administering an effective amount of a therapeuticcompound of the present invention to a subject with cancer.

In one embodiment, the present invention provides a method of treatingcancer comprising administering an effective amount of a therapeuticcompound of the present invention to a subject with cancer wherein thecancer comprises cancer stem cells.

In another embodiment, the present invention provides a method oftreating cancer comprising administering an effective amount of atherapeutic compound of the present invention to a subject with cancerwherein the cancer is recurrent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Phospholipid ether (“PLE”) analogs are sequestered via lipidrafts.

FIG. 2. Preferential uptake of CLR1501 by cancer cells. Compare uptakeof CLR1501 by cancer cell lines in (A) and (C)-(F) with normal cells in(B). (A) Renal (Caki-2). (B) Normal human skin fibroblast. (C) Ovarian(OVcar-3). (D) Pancreatic (Panc-1). (E) Melanoma (A-375). (F) Prostate(PC-3).

FIG. 3. Prolonged retention of ¹³¹I-CLR1404 by human RL-251 tumorxenograft in SCID mouse.

FIG. 4. Detection of C6-glioma in rat brain using ¹²⁵I-NM404. (A)Bioscan of sham control rat brain. (B) Bioscan image of rat brain from(A) superimposed over digital photo showing background levels of¹²⁵I-NM404 in normal brain tissue. (A′) Digital photo of C6-gliomabearing rat brain 4 days post ¹²⁵I-NM404 injection. (B′) Bioscan imageof rat brain from (A′). (C′) Position and size-matched images of (A′)and (B′) superimposed to show intense localization of NM404 in tumor.(D′) H&E stained sample confirming presence of tumor.

FIG. 5. ¹²⁴I-CLR1404 uptake in a broad range of malignant tumors.(A)-(I) are rodent models with human cancer xenografts. (J)-(M) arerodent cancer models. (A) Orthotopic glioma U87 (rat). (B) ColonHCT-116. (C) Colon HT-29. Arrow indicates location of tumor. (D) BreastMDA-MB-23 1. Arrow indicates location of tumor. (E) Prostate PC-3. (F)Metastatic PC-3. (G) PC-3 tibial xenograft. (H) Pancreatic BxPC3. Lowerarrow indicates location of tumor. Upper arrow indicates livermetastasis. (I) Ewing's sarcoma. Arrow indicates location of tumor. (J)Mouse SV40 bladder. (K) Mouse Breast 4T1. (L) Mouse pancreatic c-myc.(M) Rat brain CNS-1.

FIG. 6. Detection of non-small cell lung cancer (“NSCLC”) tumors inhuman patient using ¹³¹I-CLR1404. (A) shows gamma camera images ofPatient 1 at 4 and 11 days post ¹³¹I-CLR1404 injection. Note intense andprolonged retention of CLR1404 in the NSCLC tumors (arrows). (B and C)show the location and size of focal 3 cm lesion in left lung (A) andlarge infiltrative mass in right lung (B) (arrows). (D and E) show wholebody planar nuclear medicine images of Patient 2 1, 2 and 4 days post¹³¹I-CLR1404 IV administration. (F and G) show axial (F) and coronal (G)CT scans indicating location of large 6 cm NSCLC tumor (arrows).

FIG. 7. Detection of 3 previously unknown brain tumor metastases inNSCLC patient using ¹²⁴I-CLR1404. Arrows indicate location of tumors asimaged using PET/CT after uptake of ¹²⁴I-CLR1404 by cancer cells.

FIG. 8. Detection of tumor recurrence of a right frontal falcinemetastasis using ¹²⁴I-CLR1404. (A) MRI of brain following radiosurgery.Arrow indicates lesion which was interpreted as radiation necrosis. (B)PET image using ¹²⁴I-CLR1404 shows uptake of ¹²⁴I-CLR1404 by lesion. (C)MRI of brain 8 months after stereotactic radiosurgery shows increase insize of lesion indicating possible recurrence.

FIG. 9. PLE-Paclitaxel Conjugates IC₅₀ for MDA-MB-468. (A) freepaclitaxel, (B) CLR1601 and (C) CLR1603.

FIG. 10. PLE-Paclitaxel Conjugates IC₅₀ for NCI-H1299. (A) freepaclitaxel, (B) CLR1601 and (C) CLR1603.

FIG. 11. PLE-Paclitaxel Conjugates IC₅₀ for NCI-H460. (A) freepaclitaxel, (B) CLR1601 and (C) CLR1603.

FIG. 12. PLE-Paclitaxel Conjugates IC₅₀ for Capan-2. (A) freepaclitaxel, (B) CLR1601 and (C) CLR1603.

FIG. 13. PLE-Paclitaxel Conjugates IC₅₀ for MiaPaCa-1. (A) freepaclitaxel, (B) CLR1601 and (C) CLR1603.

FIG. 14. PLE-Paclitaxel Conjugates IC₅₀ for HT29. (A) free paclitaxel,(B) CLR1601 and (C) CLR1603.

FIG. 15. PLE-Paclitaxel Conjugates IC₅₀ for HCT116. (A) free paclitaxel,(B) CLR1601 and (C) CLR1603.

FIG. 16. PLE-Paclitaxel Conjugates IC₅₀ for PC-3. (A) free paclitaxel,(B) CLR1601 and (C) CLR1603.

FIG. 17. A representative scatter plot of flow cytometry results forMDA-MB-468 cells treated with 5 μM of CLR1601 for 72 hours. Annexin V(attached to AlexaFluor 488) is shown on the x-axis, whilephosphatidylinositide (“PI”) is shown on the y-axis. The lower leftquadrant indicates live cells, the upper left quadrant indicatesnecrotic cells, the upper right quadrant indicates late apoptotic cellsand the lower right quadrant indicates early apoptotic cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “treating” includes preventative as well asdisorder remittent treatment including reducing, suppressing andinhibiting cancer progression or recurrence. As used herein, the terms“reducing”, “suppressing” and “inhibiting” have their commonlyunderstood meaning of lessening or decreasing. As used herein, the term“progression” means increasing in scope or severity, advancing, growingor becoming worse. As used herein, the terms “recurrence” and“recurrent” refer to the return of a disease after a remission.

As used herein, the term “administering” refers to bringing a patient,tissue, organ or cells in contact with an anti-cancer compound of thepresent invention. As used herein, administration can be accomplished invitro (i.e. in a test tube) or in vivo, (i.e. in cells or tissues ofliving organisms, for example, humans). In certain embodiments, thepresent invention encompasses administering the compounds useful in thepresent invention to a patient or subject. A “patient” or “subject”,used equivalently herein, refers to a mammal, preferably a human, thateither: (1) has a disorder remediable or treatable by administration ofthe anti-cancer substance using a PLE compound or (2) is susceptible toa disorder that is preventable by administering the anti-cancer compoundof the present invention.

As used herein, the term “effective amount” refers to an amountsufficient to affect a desired biological effect, such as a beneficialresult, including, without limitation, prevention, diminution,amelioration or elimination of signs or symptoms of a disease ordisorder. Thus, the total amount of each active component of thepharmaceutical composition or method is sufficient to show a meaningfulsubject benefit. Thus, an “effective amount” will depend upon thecontext in which it is being administered. An effective amount may beadministered in one or more prophylactic or therapeutic administrations.

As used herein the term “therapeutic compound” refers to any chemicalcompound capable of providing treatment for cancer.

As used herein the term “cancer” refers to any disease that results fromthe uncontrolled division of cells capable of metastasizing.

The terms “chemotherapy drug” “anti-cancer drug” and “anti-tumor drug”are used interchangeably throughout the specification.

The term “malignant tumor cell” and “cancer cell” are usedinterchangeably throughout the specification. The term “malignant tumorstem cell” and “cancer stem cell” are used interchangeably throughoutthe specification.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, from acombination of the specified ingredients in the specified amounts.

As used herein the term “A” refers to an phospholipid ether of theformula

As used herein the term “W” refers to an aryl, a C₁-C₆ alkyl, analkenyl, an optionally substituted C₃-C₆ cycloalkyl and an optionallysubstituted C₃-C₆ heterocycloalkyl.

As used herein the term “aryl” refers to an aromatic ring including aphenyl group.

As used herein the term “alkyl” refers to a branched or straight-chainalkyl consisting of a saturated hydrocarbon group of 1 to 24 carbonatoms (C₁-C₂₄) unless otherwise stated. The alkyl group can be cyclic oracyclic.

As used herein the term “alkenyl” refers to a carbon-carbon double bond.

As used herein the term “cycloalkyl” refers to a cyclic alkyl group of 3to 24 carbon atoms (C₃-C₂₄).

As used herein the term “heterocycloalkyl” refers to a cyclic group of 3to 24 atoms (C₃-C₂₄) selected from carbon, nitrogen, sulfur, phosphateand oxygen wherein at least one atom is carbon.

In general, the term “substituted,” whether preceded by the term“optionally” or not, means that one or more hydrogens of the designatedmoiety are replaced with a suitable substituent. Unless otherwiseindicated, an “optionally substituted” group may have a suitablesubstituent at each substitutable position of the group, and when morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. Combinations ofsubstituents envisioned by this invention are preferably those thatresult in the formation of stable or chemically feasible compounds.

As used herein the term “R” refers to a hydrogen (H) or an alkyl.

As used herein the term “m” refers to an integer of 12 to 24.

As used herein the term “n” refers to an integer of 0 to 6.

As used herein the term “B” refers to a linker compound. As used hereinthe term “linker compound” refers to any chemical compound or compoundscapable of forming a chemical bond with two or more other distinctchemical compounds such that all compounds form a single largercompound. In one embodiment, the linker compound is a bond. Multiplelinker compounds may be used in the formation of the larger compound. Inspecific embodiments, the term linker compound is a bond or a compoundof the formula Y—(CH₂)_(n)—Z.

As used herein the term “Y” refers to a bond, O, NH, C═O, NHSO₂O, orOC(═O)O.

As used herein the term “Z” refers to O, NH, C═O, C(═O)O, C(═O)NH, SO₂,OC(═O)OCH₂, and —S—S—.

As used herein the term “D” refers to any anti-cancer drug currentlyknown or in development.

As defined herein, the term “isomer” includes, but is not limited tooptical isomers and analogs, structural isomers and analogs,conformational isomers and analogs, and the like. In one embodiment,this invention encompasses the use of different optical isomers of thepresent invention. It will be appreciated by those skilled in the artthat the anti-cancer compounds useful in the present invention maycontain at least one steriogenic center. Accordingly, the compounds usedin the methods of the present invention may exist in, and be isolatedin, optically-active or racemic forms. Some compounds may also exhibitpolymorphism.

It is to be understood that the present invention may encompass the useof any racemic, optically-active, polymorphic, or stereroisomeric form,or mixtures thereof, which form possesses properties useful in thetreatment of cancer-related conditions described and claimed herein. Inone embodiment, the anti-cancer compounds may include pure (R)-isomers.In another embodiment, the anti-tumor compounds may include pure(S)-isomers. In another embodiment, the compounds may include a mixtureof the (R) and the (S) isomers. In another embodiment, the compounds mayinclude a racemic mixture comprising both (R) and (S) isomers. It iswell known in the art how to prepare optically-active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase).

The invention includes the use of pharmaceutically acceptable salts ofamino-substituted compounds with organic and inorganic acids, forexample, citric acid and hydrochloric acid. The invention also includesN-oxides of the amino substituents of the compounds described herein.Pharmaceutically acceptable salts can also he prepared from the phenoliccompounds by treatment with inorganic bases, for example, sodiumhydroxide. Also, esters of the phenolic compounds can be made withaliphatic and aromatic carboxylic acids, for example, acetic acid andbenzoic acid esters. As used herein, the term “pharmaceuticallyacceptable salt” refers to a compound formulated from a base compoundwhich achieves substantially the same pharmaceutical effect as the basecompound.

This invention further includes derivatives of the anti-cancercompounds. The term “derivatives” includes but is not limited to etherderivatives, acid derivatives, amide derivatives, ester derivatives andthe like. In addition, this invention further includes methods utilizinghydrates of the anti-tumor compounds. The term “hydrate” includes but isnot limited to hemihydrate, monohydrate, dihydrate, trihydrate and thelike.

This invention further includes metabolites of the anti-cancercompounds. The term “metabolite” means any substance produced fromanother substance by metabolism or a metabolic process.

Cancers that can be treated with compounds of the present inventioninclude, but are not limited to: breast cancer including male breastcancer; digestive/gastrointestinal cancers including anal cancer,appendix cancer, extrahepatic bile duct cancer, gastrointestinalcarcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer,gastric cancer, gastrointestinal stromal tumors (“gist”), Islet celltumors, adult primary liver cancer, childhood liver cancer, pancreaticcancer, rectal cancer, small intestine cancer, and stomach (gastric)cancer; endocrine and neuroendocrine cancers including pancreaticadenocarcinoma, adrenocortical carcinoma, pancreatic neuroendocrinetumors, Merkel cell carcinoma, non-small cell lung neuroendocrine tumor,small cell lung neuroendocrine tumor, parathyroid cancer,pheochromocytoma, pituitary tumor and thyroid cancer; eye cancersincluding intraocular melanoma and retinoblastoma; genitourinary cancerincluding bladder cancer, kidney (renal cell) cancer, penile cancer,prostate cancer, transitional cell renal pelvis and ureter cancer,testicular cancer, urethral cancer and Wilms tumor; germ cell cancersincluding childhood central nervous system cancer, childhoodextracranial germ cell tumor, extragonadal germ cell tumor, ovarian germcell tumor and testicular cancer; gynecologic cancers including cervicalcancer, endometrial cancer, gestational trophoblastic tumor, ovarianepithelial cancer, ovarian germ cell tumor, uterine sarcoma, vaginalcancer and vulvar cancer; head and neck cancers including hypopharyngealcancer, laryngeal cancer, lip and oral cavity cancer, metastaticsquamous neck cancer with occult primary, mouth cancer, nasopharyngealcancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer,parathyroid cancer, pharyngeal cancer, salivary gland cancer and throatcancer; leukemias including adult acute lymphoblastic leukemia,childhood acute lymphoblastic leukemia, adult acute myeloid leukemia,childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia and hairy cell leukemia; lymphomas includingAIDS-related lymphoma, cutaneous t-cell lymphoma, adult Hodgkinlymphoma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy,mycosis fungoides, adult non-Hodgkin lymphoma, childhood non-Hodgkinlymphoma, non-Hodgkin lymphoma during pregnancy, primary central nervoussystem lymphoma, Sézary syndrome and Waldenström macroglobulinemia;musculoskeletal cancers including Ewing sarcoma, osteosarcoma andmalignant fibrous histocytoma of bone, childhood rhabdomyosarcoma andsoft-tissue sarcoma; neurological cancers including adult brain tumor,childhood brain tumor, astrocytomas, brain stem glioma, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, craniopharyngioma, ependymoma, neuroblastoma, primarycentral nervous system (CNS) lymphoma; respiratory/thoracic cancersincluding non-small cell lung cancer, small cell lung cancer, malignantmesothelioma, thymoma and thymic carcinoma; and skin cancers includingKaposi sarcoma, melanoma and squamous cell carcinoma.

Compounds of formula (I) of the present invention have been demonstratedto be sequestered by cancer stem cells. See, Weichert J.P., et al.(2014) at 2, FIG. 2 (demonstrating that CLR-1501, a CLR1404 fluorescentanalog, has enhanced uptake by human glioblastoma stemlike cells andserum-cultured human glioblastoma cells as compared to normal humanastrocytes and fetal human neural stem cells.) Cancer stem cells areassociated with most, if not all, major cancer types. Tumor hypoxiastimulates cancer stem cell propagation, leading to increased resistanceand metastatic potential. As such, cancer stem cells are associated withchemotherapy resistance, tumor re-growth, and metastasis followingchemotherapy and radiation therapy. Thus, compounds of the presentinvention have the potential to treat various forms of cancer that haveproven resistant to traditional therapy regimens.

Compounds of the Invention

Drug delivery vehicles that are useful for the present inventioninclude, but are not limited to, compounds of formula (I),

or formula (II),

or formula (III)

or a combination thereof, wherein W is selected from the groupconsisting of an aryl, a C₁-C₆ alkyl, an alkenyl, an optionallysubstituted C₃-C₆ cycloalkyl and an optionally substituted C₃-C₆heterocycloalkyl, wherein R is H or an alkyl and wherein m is an integerfrom 12 to 24.

The basis for selective tumor targeting of compounds of the presentinvention lies in differences between the plasma membranes of cancercells as compared to those of most normal cells. Specifically, cancercell membranes are highly enriched in “lipid rafts”. Cancer cells havefive to ten times more lipid rafts than healthy cells. Lipid rafts arespecialized regions of the membrane phospholipid bilayer that containhigh concentrations of cholesterol and sphingolipids and serve toorganize cell surface and intracellular signaling molecules (e.g.,growth factor and cytokine receptors, the phosphatidylinositol 3-kinase(PI3K)/Akt survival pathway). Data suggests that lipid rafts serve asportals of entry for PLEs. The marked selectivity of these compounds forcancer cells versus non-cancer cells is attributed to the high affinityof PLEs for cholesterol and the abundance of cholesterol-rich lipidrafts in cancer cells. The pivotal role played by lipid rafts isunderscored by the fact that disruption of lipid raft architecturesuppresses uptake of PLEs into cancer cells. It has been shown that theuptake of PLE's is reduced by 60% when lipid rafts are blocked fromforming. (See Example 2 and FIG. 1).

Preliminary results obtained in over 55 xenograft and spontaneous tumormodels have universally shown CLR1404 to undergo selective uptake andprolonged retention in tumors. Because the agent is metabolized to someextent in the liver, the inventors avoided earlier compound evaluationin liver tumor models due to high liver background radioactivity levels.

CLR1404 is a PLE. Results obtained in a variety of tumor models indicatethat CLR1404 is sequestered and selectively retained by cancer cells andcancer stem cells. In fact, CLR1404 has been shown to remain in cancercells for up to 20 days. See FIG. 3. CLR1404 localizes in both primaryand metastatic lesions regardless of anatomic location including thosefound in lymph nodes. See Examples 3-8. The high tumor to backgroundavidity and tumor selectivity of CLR1404 suggests the core molecule iswell-suited for use as an anti-cancer drug delivery vehicle.

Linker compounds that are useful for the present invention include anychemical linker capable of binding a drug delivery vehicle of thepresent invention to an anti-cancer drug of the present invention.Linker compounds that are useful for the present invention include bothcleavable and non-cleavable linkers. In one embodiment, linker compoundsthat are useful for the present invention include, but are not limitedto, aminobutyramide, amino acids, glutaramic acids, dicarboxylic acids,carbamic acids, a carbonyl, 9,10-anthracenedicarboxylic acid,biphenyl-3,3′,5,5′-tetracarboxylic acid, biphenyl-3,4′,5-tricarboxylicacid, 5-bromoisophthalic acid, 5-cyano-1,3-benzenedicarboxylic acid,2,2′-diamino-4,4′-stilbenedicarboxylic acid, 2,5-diaminoterephthalicacid, 2,5-dihydroxyterephthalic acid, 5-ethynyl-1,3-benzenedicarboxylicacid, 2-hydroxyterephthalic acid, imidazole, 2-methylimidazole,2,6-naphthalenedicarboxylic acid, oxalic acid dehydrate, terephthalicacid, [1,1′:4′,1″]terphenyl-3,3″,5,5″-tetracarboxylic acid,3,3′,5,5′-tetracarboxydiphenylmethane,1,2,4,5-tetrakis(4-carboxyphenyl)benzene,4,4′,4″-s-triazine-2,4,6-triyl-tribenzoic acid, trimesic acid,1,3,5-tris(4′-carboxy[1,1′-biphenyl]-4-yl)benzene,1,3,5-tris(4-carboxyphenyl)benzene, and 1,3,5-triscarboxyphenylethynylbenzene.

In another embodiment, linker compounds useful for the present inventionalso include, but are not limited to, lysosomal protease sensitivelinkers with or without an aniline-based self-immolative fragment.Non-limiting examples of lysosomal protease sensitive linkers are

which contain the valine-citrulline dipeptide linker designed to displayan optimal balance between plasma stability and intracellular proteasecleavage. See, Tranoy-Opalinski I., Design of self-immolative linkersfor tumour-activated prodrug therapy, Anticancer Agents Med Chem, 2008Aug. 8(6):618-637, which is incorporated by reference herein in itsentirety.

In another embodiment, linker compounds useful for the present inventionalso include, but are not limited to, self-immolative linkers that arecleaved by β-glucuronidase. β-glucuronidase is present in highconcentration in necrotic area surrounding cancer cells. See,Tranoy-Opalinski I., β-glucuronidase-responsive prodrugs for selectivecancer chemotherapy: An update, Eur J Med Chem, 2014 Mar. 3, 74,302-313, which is incorporated by reference herein in its entirety.Non-limiting examples of β-glucuronidase-cleavable self-immolativelinkers are

wherein X is NH₂ or NO₂ and wherein Y is O or NCH_(3.)

Preferred linker compounds of the present invention are a bond or acompound of formula (IV), Y—(CH₂)_(n)—Z (IV), wherein:

-   -   Y is bound to A;    -   Z is bound to D;    -   Y is selected from the group consisting of a bond, O, NH, C═O,        NHSO₂O, and OC(═O)O; and    -   Z is selected from the group consisting of O, NH, C═O, C(═O)O,        C(═O)NH, SO₂, OC(═O)OCH₂, and —S—S—; and    -   n is an integer from 0 to 6.

More preferred linker compounds of the present invention are a bond or acompound of formula (IV), wherein n is an integer from 0 to 6, Y isbound to A, Z is bound to D, Y is selected from the group consisting ofa bond and C═Oand Z is selected from the group consisting of NH, C═O,C(═O)NH and C(═O)O.

Anti-cancer drugs that are useful for the present invention include, butare not limited to, paclitaxel, irinotecan, topotecan, gemcitabine,cisplatin, geldanamycin, mertansine, abiraterone, afatinib,aminolevulinic acid, aprepitant, axitinib, azacitidine, belinostat,bendamustine, bexarotene, bleomycin, bortezomib, bosutinib, busulfan,cabazitaxel, cab ozantinib, capecitabine, carboplatin, carfilzomib,carmustine, ceritinib, cetuximab, chlorambucil, clofarabine, crizotinib,cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin,dasatinib, daunorubicin, decitabine, denosumab, dexrazoxane, docetaxel,dolastatins (e.g. monomethyl auristatin E), doxorubicin, enzalutamide,epirubicin, eribulin mesylate, erlotinib, etoposide, everolimus,floxuridine, fludarabine phosphate, fluorouracil, ganetespib, gefitinib,gemtuzumab ozogamicin, hexamethylmelamine, hydroxyurea, ibritumomabtiuxetan, ibrutinib, idelalisib, ifosfamide, imatinib, ipilimumab,ixabepilone, lapatinib, leucovorin calcium, lomustine, maytansinoids,mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate,mitomycin C, mitotane, mitoxantrone, nelarabine, nelfinavir, nilotinib,obinutuzumab, ofatumumab, omacetaxine mepesuccinate, oxaliplatin,panitumumab, pazopanib, pegaspargase, pembrolizumab, pemetrexed,pentostatin, pertuzumab, plicanycin, pomalidomide, ponatinibhydrochloride, pralatrexate, procarbazine, radium 223 dichloride,ramucirumab, regorafenib, retaspimycin, ruxolitinib, semustine,siltuximab, sorafenib, streptozocin, sunitinib malate, tanespimycin,temozolomide, temsirolimus, teniposide, thalidomide, thioguanine,thiotepa, toremifene, trametinib, trastuzumab, vandetanib, vemurafenib,vinblastine, vincristine, vinorelbine, vismodegib, vorinostat, andziv-aflibercept. Any compounds that are currently known to or arecapable of acting as anti-cancer drugs are also useful for the presentinvention.

PLE drug delivery vehicles of the present invention may attachsingularly or in multiple to an anti-cancer drug in any number ofpossible stable attachment sites via a linker compound or directly.

Compositions of the Invention

In another aspect, the present invention provides a pharmaceuticalcomposition containing a compound of the present invention incombination with one or more pharmaceutically acceptable carriers. In apreferred aspect the pharmaceutical composition is free of Kolliphor® EL(Kolliphor is a registered trademark of BASF SE). Kolliphor® EL isformerly known as Cremophor® EL (Cremophor is a registered trademark ofBASF SE).

Actual dosage levels of active ingredients in the therapeuticcompositions of this invention can be varied so as to obtain an amountof the active compound(s) which is effective to achieve the desiredtherapeutic response for a particular patient, compositions and mode ofadministration. The selected dosage level will depend upon the activityof the particular compound, the route of administration, the severity ofthe condition being treated and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved.

The phrase “therapeutically effective amount” of the compound of theinvention means a sufficient amount of the compound to treat disorders,at a reasonable benefit/risk ratio applicable to any medical treatment.It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds of this invention administered toa human or lower animal may range from about 0.0001 to about 1000mg/kg/day. For purposes of oral administration, more preferable dosescan be in the range of from about 0.001 to about 5 mg/kg/day. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration; consequently, single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.

The present invention also provides pharmaceutical compositions thatcomprise compounds of the present invention formulated together with oneor more pharmaceutically acceptable carriers. The pharmaceuticalcompositions can be specially formulated for oral administration insolid or liquid form, for parenteral administration or for rectaladministration.

The pharmaceutical compositions of this invention can be administered tohumans and other mammals orally, rectally, parenterally,intracisternally, intravaginally, transdermally (e.g. using a patch),transmucosally, sublingually, pulmonary, intraperitoneally, topically(as by powders, ointments or drops), bucally or as an oral or nasalspray. The terms “parenteral” or “parenterally,” as used herein, refersto modes of administration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a component of the present invention and aphysiologically tolerable diluent. The present invention includes one ormore compounds as described above formulated into compositions togetherwith one or more physiologically tolerable or acceptable diluents,carriers, adjuvants or vehicles that are collectively referred to hereinas diluents, for parenteral injection, for intranasal delivery, for oraladministration in solid or liquid form, for rectal or topicaladministration, among others.

Compositions suitable for parenteral injection may comprisephysiologically acceptable, sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), vegetable oils (such asolive oil), injectable organic esters such as ethyl oleate, and suitablemixtures thereof.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound may be mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier, such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol and silicic acid; b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcoholand glycerol monostearate; h) absorbents such as kaolin and bentoniteclay and i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well-known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and may also be of acomposition such that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof.

Besides inert diluents, the oral compositions may also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring and perfuming agents.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active compound.

Compounds of the present invention can also be administered in the formof liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multi-lamellar hydrated liquid crystals which are dispersed inan aqueous medium. Any, physiologically acceptable and metabolizablelipid capable of forming liposomes can be used. The present compositionsin liposome form can contain, in addition to a compound of the presentinvention, stabilizers, preservatives, excipients and the like. Thepreferred lipids are natural and synthetic phospholipids andphosphatidyl cholines (lecithins) used separately or together. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq. Such compositions will influence the physicalstate, solubility, stability, rate of in vivo release, and rate of invivo clearance.

In one method of the present invention, a pharmaceutical composition canbe delivered in a controlled release system. For example, the agent maybe administered using intravenous infusion, an implantable osmotic pump,a transdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment,polymeric materials can be used. In yet another embodiment, a controlledrelease system can be placed in proximity to the therapeutic target, forexample liver, thus requiring only a fraction of the systemic dose (see,e.g., Goodson, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138 (1984). Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533 (1990).

In another aspect, the invention is directed to a method of treating adisease or condition in a subject comprising administering to thesubject an effective amount of a compound of the present invention.

In general, the invention is not limited to treatment of any specificdisease or condition but encompasses the treatment of any disease orcondition whose mechanism may be affected by the compounds of thepresent invention.

Representative Embodiments

Paclitaxel-CLR1404 Conjugates

In one embodiment of the present invention the therapeutic compound ispaclitaxel linked to an CLR1404 core compound by a dicarboxylic acidlinker, wherein the dicarboxylic acid linker is attached to the CLR1404core compound via an amide bond and to paclitaxel via an ester bond atthe 2′-OH group,

In a preferred embodiment of the present invention the therapeuticcompound is paclitaxel linked to the CLR1404 core compound by aglutaramic acid linker, wherein the glutaramic acid linker is attachedto the CLR1404 core compound via an amide bond and to paclitaxel via anester bound at the 2′-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to the CLR1404 core compound by a dicarboxylic acidlinker, wherein the dicarboxylic acid linker is attached to the CLR1404core compound via an amide bond and to paclitaxel via an ester bond atthe 7-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to the CLR1404 core compound by a carbamic acidlinker, wherein the carbamic acid linker is attached to the CLR1404 corecompound via an amide bond and to paclitaxel via an ester bond at the7-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to the CLR1404 core compound by acarbonic-carboxylic acid linker, wherein the carbonic-carboxylic acidlinker is attached to the CLR1404 core compound via an amide bond and topaclitaxel via an ester bond at the 7-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to two CLR1404 core compounds by dicarboxylic acidlinkers, wherein the dicarboxylic acid linkers are attached to theCLR1404 core compounds via amide bonds and to paclitaxel via ester bondsat both the 2′-OH group and the 7-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to the CLR1404 core compound by a dicarboxylic acidlinker, wherein the dicarboxylic acid linker is attached to the CLR1404core compound via an amide bond and to paclitaxel via a carbonate or acarbamate bond at the 2′-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to an CLR1404 core compound by a dicarboxylic acidlinker, wherein the dicarboxylic acid linker is attached to the CLR1404core compound via an amide bond and to paclitaxel via a carbonate or acarbamate bond at the 7-OH group,

In another embodiment of the present invention the therapeutic compoundis paclitaxel linked to two CLR1404 core compounds by dicarboxylic acidlinkers, wherein the dicarboxylic acid linkers are attached to the twoCLR1404 core molecules via amide bonds and to paclitaxel via a carbonateor a carbamate bond at both the 2′-OH group and the 7-OH group,

In one embodiment of the present invention the therapeutic compound ispaclitaxel linked to a C18 alkyl phosphocholine compound via acarboxylic linker, wherein the carboxylic linker is attached to the C18alkyl phosphocholine compound via an amide or carbonate bond and topaclitaxel via a carbonate or a carbamate bond at the 2′-OH group

Irinotecan-CLR1404 Conjugate

In one embodiment of the present invention the therapeutic compound isirinotecan linked to the CLR1404 compound by a dicarboxylic acid linker,wherein the dicarboxylic acid linker is attached to the CLR1404 corecompound via an a carbonate or a carbamate bond and to irinotecan via anester bond,

Irinotecan-C18 Alkyl Phosphocholine Conjugate

In one embodiment of the present invention the therapeutic compound isirinotecan linked to a C18 alkyl phosphocholine compound by a carbonyllinker, wherein the carbonyl linker is attached to the C18 alkylphosphocholine compound via a carbon-carbon bond and to irinotecan viaan ester bond,

Topotecan-CLR1404 Conjugates

In one embodiment of the present invention the therapeutic compound istopotecan linked to the CLR1404 core compound by a non-hydrolyzablephenyl ether,

In another embodiment of the present invention the therapeutic compoundis topotecan linked to an CLR1404 core compound by a dicarboxylic acidlinker, wherein the dicarboxylic acid linker is attached to the CLR1404compound via a carbonate or a carbamate bond and to topotecan via anester bond,

Gemcitabine-C18 Alkyl Phosphocholine Conjugate

In one embodiment of the present invention the therapeutic compound isgemcitabine linked to two C18 alkyl phosphocholine compounds by carbonyllinkers, wherein the carbonyl linkers are attached to the C18 alkylphosphocholine compounds via carbon-carbon bonds and to gemcitabine viaester bonds,

In another embodiment of the present invention the therapeutic compoundis gemcitabine linked to a C18 alkyl phosphocholine compound by acarbonyl linker, wherein the carbonyl linker is attached to the C18alkyl phosphocholine compound via a carbon-carbon bond and togemcitabine via an ester bond,

Cisplatin-CLR1404 Core Conjugate

In one embodiment of the present invention the therapeutic compound iscisplatin linked directly to the CLR1404 core compound,

Geldanamycin-CLR1404 Conjugates

In one embodiment of the present invention the therapeutic compound isgeldanamycin linked directly to the CLR1404 core comnound

In another embodiment of the present invention the therapeutic compoundis geldanamycin linked to the CLR1404 core compound by a short aminoacid linker, wherein the amino acid linker is connected to the CLR1404core compound via a carbonate or carbamate bond and to the geldanamycinvia an amide bond,

In a preferred embodiment of the present invention the therapeuticcompound is geldanamycin linked to the CLR1404 core compound by anaminobutyramide linker, wherein the aminobutyramide linker is connectedto the CLR1404 core compound via a carbamate bond and to thegeldanamycin via an amide bond:

Mertansine-CLR1404 Conjugates

In another embodiment of the present invention the therapeutic compoundis mertansine linked to the CLR1404 core compound by a maleimide linker,wherein the maleimide linker is attached to the CLR1404 core compoundvia an amide bond and to mertansine via a carbon-sulfur bond,

For all representative embodiments n is an integer from 2 to 6 and X isO or NH.

EXAMPLES Example 1 Syntheses of Conjugates

I. Synthesis of CLR1601

A. Synthesis of CLR1401 azide

18-(p-Iodophenyl)octadecyl phosphocholine (4.01 g, 6.3 mmol), sodiumazide (818 mg, 12.6 mmol) and sodium ascorbate (140 mg, 0.71 mmol) weredissolved in the mixture of degassed ethanol (28 ml) and water (12 ml)in the reaction vessel. Copper (I) iodide (120 mg, 0.63 mmol) andN,N′-dimethyl-ethylenediamine (0.1 ml, 0.94 mmol) were added to thereaction mixture. Reaction vessel was tightly closed and the mixture wasstirred at 80° C. for 45 min. Reaction mixture was cooled to the roomtemperature, water (60 ml) was added, and the mixture was stirred for 30min open to the air. The mixture was transferred to the separatoryfunnel, chloroform (80 ml) and methanol (52 ml) were added, andextraction was performed by shaking. Chloroform layer was removed, andextraction was repeated (2×80 ml of chloroform). Combined chloroformextracts were washed with 0.01 N HCl, dried over Na₂SO₄, filtered andevaporated to dryness. Residue was dissolved in chloroform (4 ml) andacetone (170 ml) was slowly added with stirring. The mixture was stirredfor 30 min and filtered. The product was rinsed on the filter withacetone, and dried under high vacuum to give 3.31 g (95%) of18-(p-azidophenyl)octadecyl phosphocholine.

B. Synthesis of CLR1401 amine

18-(p-Azidophenyl)octadecyl phosphocholine (3.116 g) was placed in aParr pressure bottle, methanol (30 ml) and catalyst 10% Pd/C (100 mg)were added. The hydrogenation reaction was performed under hydrogenpressure (55 psi) with shaking for 24 h. The bottle was depressurized,chloroform and methanol were added to dissolve some precipitatedreaction product, and the mixture was filtered to remove the catalyst.Filtrate was evaporated to dryness and residue was dissolved in warmchloroform-methanol (1:1) mixture (10 ml). Hot acetone (150 ml) wasadded slowly with stirring, the mixture was cooled to the ambienttemperature with stirring and filtered. Product was rinsed on the filterwith acetone and dried under high vacuum. Yield of18-(p-aminophenyl)octadecyl phosphocholine: 2.597 g (87%).

C. Synthesis of paclitaxel-2′-hemiglutarate

Paclitaxel (404 mg, 0.437 mmol) and glutaric anhydride (67 mg, 0.588mmol) were dissolved in chloroform (8 ml) and pyridine (0.5 ml) wasadded. Reaction mixture was stirred at room temperature for 24 h andevaporated to dryness. Residue was kept under high vacuum for 1.5 h toremove the residual pyridine. Crude product was purified by silica gelchromatography in chloroform-methanol (gradient from 98:2 to 95:5) toyield 452 mg (99%) of paclitaxel-2′-hemiglutarate.

D. Synthesis of CLR1601

Paclitaxel-2′-hemiglutarate (947 mg, 0.978 mmol) and18-(p-aminophenyl)octadecyl phosphocholine (492 mg, 0.934 mmol) weresuspended in chloroform (40 ml) and isopropanol (1.2 ml) mixture. Tothis suspension, trimethylamine (0.27 ml, 1.957 mmol) and COMU (419 mg,0.978 mmol) were added. Reaction mixture was stirred at room temperaturefor 20 h by which time it became clear and homogeneous. Reaction mixturewas transferred to a separation funnel and mixed with chloroform (40ml), methanol (80 ml) and cold water (72 ml). Chloroform layer wasremoved, and extraction was repeated (2×80 ml of chloroform). Combinedchloroform extracts were dried over Na₂SO₄, filtered and evaporated todryness. The remaining residue was purified by chromatography on silicagel with chloroform-methanol (gradient from 9:1 to 5:5) followed byfinal elution with chloroform-methanol-water (65:25:4). Afterevaporation of the solvent, the product was dried under high vacuum togive 1.167 g (85%) of CLR1601.

II. Synthesis of CLR1602

A. Synthesis of 18-[p-(4-N-BOC-aminobutyramido)phenyl]octadecylphosphocholine

18-(p-Aminophenyl)octadecyl phosphocholine (76 mg, 0.144 mmol) and4-N-BOC-aminobutyric acid (38 mg, 0.188 mmol) were suspended inchloroform (5 ml) and isopropanol (0.15 ml), then triethylamine (0.05ml, 0.38 mmol) was added followed by COMU (80 mg, 0.188 mmol). Reactionmixture was stirred at room temperature for 24 h and quenched with 2 mlof saturated aqueous NaHCO₃ solution. Quenched reaction mixture wastransferred into to a separation funnel and mixed with chloroform (35ml), methanol (40 ml) and cold water (36 ml). Chloroform layer wasremoved, and extraction was repeated (2×40 ml of chloroform). Combinedchloroform extracts were dried over Na₂SO₄, filtered and evaporated todryness. Residue was purified by chromatography on silica gel withchloroform-methanol (gradient from 9:1 to 5:5) followed by final elutionwith chloroform-methanol-water (65:25:4). After evaporation of thesolvent, the product was dissolved in warm chloroform-methanol mixture(1.5 ml) and precipitated with acetone. Product was collected byfiltration and drying under high vacuum to give a white powder (100 mg,97%).

B. Synthesis of 18-[p-(4-aminobutyramido)phenyl]octadecyl phosphocholine

18-[(4-N-BOC-aminobutyramido) phenyl]octadecyl phosphocholine (98 mg,0.138 mmol) was dissolved in a mixture of chloroform (4 ml), methanol (2ml) and concentrated HCl (0.2 ml). The reaction mixture was stirredovernight at ambient temperature and then was quenched by slow additionof the saturated aqueous NaHCO₃ solution (3 ml). Quenched reactionmixture was transferred into a separation funnel and mixed withchloroform (40 ml), methanol (40 ml) and cold water (36 ml). Chloroformlayer was removed, and extraction was repeated (2×40 ml of chloroform).Combined chloroform extracts were dried over Na₂SO₄, filtered andevaporated to dryness. Product was purified by chromatography on silicagel with chloroform-methanol (100:65) followed by final elution withchloroform-methanol-conc. NH₄OH(aq) (100:65:15). After evaporation ofthe solvent, the product was dried under high vacuum to afford 50 mg(60%) of 18-[p-(4-aminobutyramido) phenyl]octadecyl phosphocholine.

C. Synthesis of 7-(p-nitrophenyl carbonate) paclitaxel

Paclitaxel (100 mg, 0.117 mmol) was dissolved in chloroform (4.5 ml), 8drops of pyridine were added and the solution was cooled in an ice bath.Solid p-nitrophenyl chloroformate (200 mg, 1 mmol) was added in oneportion. Reaction mixture was allowed to warm to ambient and was stirredfor 24 h, then quenched with water (1 ml) and stirred for 15 min. Themixture was extracted with chloroform, the extract was washed withwater, dried over Na₂SO₄, filtered and evaporated to dryness. Crudebis-2′,7-(pnitrophenyl carbonate) paclitaxel was dissolved in chloroformand loaded on the silica gel column. The crude product was left in thecolumn for 72 h to complete hydrolysis of p-nitrophenyl carbonate at2′-position. The column was eluted with dichloromethane—ethyl acetate(gradient from 98:2 to 90:10). After evaporation of the solvent, theproduct was precipitated with hexane and dried under high vacuum toprovide 63 mg (53%) of 7-(p-nitrophenyl carbonate) paclitaxel. See.Arpicco S., et al., Int J Pharm, 2013, 454, 653-659.

D. Synthesis of CLR1602

7-(p-Nitrophenyl carbonate) paclitaxel (53 mg, 0.052 mmol) and18-[p-(4-aminobutyramido) phenyl]octadecyl phosphocholine (47 mg, 0.077mmol) were suspended in chloroform (2 ml) and pyridine (0.5 ml) andstirred at 40° C. for 5 h. The reaction mixture was evaporated todryness, and residue was purified by chromatography on silica gel withchloroform-methanol (gradient from 9:1 to 5:5) followed by final elutionwith chloroform-methanol-water (65:25:4). After evaporation of thesolvent, compound was dried under high vacuum to give 67 mg (86%) ofsolid CLR1602.

III. Synthesis of CLR1603

A. Synthesis of 18-[p-(5-benzyloxy-valeramido)phenyl]octadecylphosphocholine

18-(p-Aminophenyl)octadecyl phosphocholine (760 mg, 1.443 mmol) and5-benzyloxyvaleric acid (361 mg, 1.732 mmol; synthesized according toCan J Chem, 1992, 70, 1472-1445 and Org Lett, 2014, 16, 516-519) weresuspended in chloroform (25 ml) and triethylamine (0.3 ml, 2.164 mmol)was added followed by solid COMU (741 mg, 1.732 mmol). Reaction mixturewas stirred at room temperature for 24 h and after completion, it wastransferred into a separation funnel and mixed with chloroform (55 ml),methanol (80 ml) and cold water (72 ml). Chloroform layer was removed,and extraction was repeated (2×80 ml of chloroform). Combined chloroformextracts were dried over Na₂SO₄, filtered and evaporated to dryness.Residue was purified by chromatography on silica gel withchloroform-methanol (gradient from 9:1 to 5:5) followed by final elutionwith chloroform-methanol-water (65:25:3). After evaporation of thesolvent and drying under high vacuum, the product was dissolved in warmchloroform-methanol mixture (3 ml) and hot acetone (75 ml) was slowlyadded with stirring. The mixture was cooled to the ambient temperaturewith stirring and filtered. Collected product was dried under highvacuum to give 18-[p-(5-benzyloxy-valeramido)phenyl]octadecylphosphocholine (887 mg, 86%) as a white powder.

B. Synthesis of 18-[p-(hydroxy-valeramido)phenyl]octadecylphosphocholine

18-[p-(5-Benzyloxy-valeramido)phenyl]octadecyl phosphocholine (868 g)was dissolved in methanol (15 ml), transferred into a Parr pressurebottle, and 10% Pd/C (75 mg) catalyst was added. The hydrogenationreaction was performed under hydrogen pressure (55 psi) with shaking for24 h. The bottle was depressurized, and the mixture was filtered toremove the catalyst. Filtrate was evaporated to dryness and residue wasdissolved in warm chloroform-methanol mixture (3-4 ml). Hot acetone (75ml) was slowly added with stirring. The mixture was cooled to theambient temperature with stirring and filtered. Collected product wasdried under high vacuum to yield18-[p-(5-hydroxy-valeramido)phenyl]octadecyl phosphocholine (718 mg,95%) as a white powder.

C. Synthesis of18-[p-(5-(p-nitro-phenoxycarbonyloxy)valeramido)phenyl]octadecylphosphocholine

18-[p-(5-Hydroxy-valeramido)phenyl]octadecyl phosphocholine (40 mg,0.064 mmol) and p-nitrophenyl chloroformate (25 mg, 0.124 mmol) weresuspended in chloroform (3 ml) and pyridine (0.2 ml) was added. Thereaction mixture was stirred for 24 h at room temperature. An additionalportion of p-nitrophenyl chloroformate (15 mg) was added, and stirringwas continued for another 1.5 h. Reaction was complete by TLC analysis.Reaction mixture was quenched with 1 ml of 1N HCl and transferred intothe separation funnel with chloroform (20 ml), methanol (20 ml) and coldwater (15 ml). Extraction was repeated (3×20 ml of chloroform). Combinedchloroform extracts were dried over Na₂SO₄, filtered and evaporated todryness. Residue was purified by chromatography on silica gel withchloroform-methanol (gradient from 9:1 to 5:5) followed by final elutionwith chloroform-methanol-water (65:25:4). After evaporation of solventand precipitation with acetone, the residue was dried under high vacuumto give 48 mg (95%) of solid material.

D. Synthesis of CLR1603

Paclitaxel (46 mg, 0.054 mmol) and18-[p-(5-(p-nitro-phenoxycarbonyloxy)valeramido)phenyl]octadecylphosphocholine (43 mg, 0.054 mmol) were suspended in chloroform (2 ml)and pyridine (0.5 ml) in a reaction vial. DMAP (8 mg, 0.065 mmol) wasadded, the vial was tightly closed and the contents were stirred at 60°C. for 48 h. An additional quantity of paclitaxel (20 mg) was added, andthe reaction was continued at 60° C. for another 48 h. Reaction mixturewas concentrated, and residue was purified by silica gel chromatographywith chloroform-methanol (gradient from 9:1 to 5:5) followed by finalelution with chloroform-methanol-water (65:25:2) and (65:25:4).Evaporation of solvent and drying under high vacuum provided CLR1603 (50mg, 62%).

IV. Synthesis of CLR1607

Geldanamycin (111 mg, 0.198 mmol) and 18-[p-(4-aminobutyramido)phenyl]octadecyl phosphocholine (110 mg, 0.18 mmol) were dissolved inchloroform (3.5 ml) and methanol (1 ml). One drop of triethylamine wasadded, and the reaction mixture was stirred at room temperature for 24h. TLC showed about 80% completion of reaction. Additional geldanamycin(10 mg) was added, and stirring was continued for another 24 h. Reactionmixture was concentrated and residue purified by silica gelchromatography with chloroform-methanol (gradient from 9:1 to 5:5)followed by final elution with chloroform-methanol-water (65:25:2),(65:25:3) and (65:25:4). After evaporation of the solvent and dryingunder high vacuum, acetone was added and the mixture was evaporated.CLR1607 was obtained as a purple solid (174 mg, 85%).

Identity for each isolated product was confirmed by ¹H-nmr and massspectral analysis.

Examples 2 through 8 exhibit the ability of CLR1404 and relatedmolecules to be sequestered and retained by various cancer types whilesimultaneously being eliminated from healthy tissue.

V. Synthesis of CLR1608

A. Synthesis of CLR1401 Maleamic Acid

CLR1401 amine (300 mg, 0.57 mmol) was dissolved in N,N-dimethylacetamide(12 ml) at 90° C. and maleic anhydride (61 mg, 0.627 mmol) was added inone portion. Reaction mixture was stirred at 90° C. for 1 h, cooled tothe room temperature and stirred for 24 h. Acetone (25 ml) was slowlyadded with stirring, and the mixture was stirred at room temperature for1 h. Precipitated product was filtered and rinsed on the filter withacetone, then dried under high vacuum. Yield: 327 mg (92%).

B. Synthesis of CLR1401 Maleimide

CLR1401 maleamic acid (100 mg, 0.16 mmol) was suspended in ethanol-freechloroform (5 ml), then triethylamine (0.05 ml, 0.352 mmol) and COMU (75mg, 0.176 mmol) were added. The reaction mixture was stirred for 24 h,then transferred to a separation funnel and mixed with chloroform (40ml), methanol (40 ml) and cold water (36 ml). Chloroform layer wasremoved, and extraction was repeated (2×40 ml of chloroform). Combinedchloroform extracts were dried over Na₂SO₄, filtered and evaporated todryness. The remaining residue was purified by chromatography on silicagel with chloroform-methanol (gradient from 9:1 to 5:5) followed byfinal elution with chloroform-methanol-water (65:25:4). Afterevaporation of the solvent, the product was precipitated with acetone,collected and dried under high vacuum to give 87 mg (90%) of CLR1401maleimide.

C. Synthesis of CLR1608

CLR1401 maleimide (40 mg, 0.066 mmol) and mertansine (53 mg, 0.072 mmol)were dissolved in the mixture of chloroform (1.7 ml) and methanol (0.3ml). Triethylamine (0.08 ml) was added, and the mixture was stirred at37° C. for 24 h. The reaction mixture was concentrated, and residue waspurified by chromatography on silica gel with chloroform-methanol(gradient from 9:1 to 5:5) followed by final elution withchloroform-methanol-water (65:25:3). After evaporation of the solvent,the product was dried under high vacuum to give 62 mg (70%) of CLR1608.

Example 2 CLR1501 is Preferentially Sequestered by Cancer Cells viaLipid Rafts

Materials and Methods:

PC-3 cells were pretreated with either 2 μg/ml filipin III or vehiclefor 15 min, then washed and incubated with 2 μCi of ¹²⁵I-CLR1404 for 1h. The media was removed and the cells were washed with phosphatebuffered saline containing 0.1% bovine serum albumin, trypsinized, thensplit into two samples for determination of cell number by DNA content(A₂₈₀ compared to a cell line specific standard curve) and counts perminute using a Gamma Counter (Perkin Elmer).

Results:

Pretreatment of PC-3 cells with filipin III, an agent that sequesterscholesterol and disrupts lipid rafts, resulted in nearly 40% less uptakeof ¹²⁵I-CLR1404 compared to untreated control cells (FIG. 1). Thissupports the hypothesis that CLR1404 uses lipid rafts as portals ofentry into cancer cells. Notably, higher filipin III concentrations arecytotoxic, and therefore, complete lipid raft ablation (and presumablycomplete inhibition of CLR1404 analog uptake) could not be demonstrated.

Example 3 Preferntial Uptake of CLR-1501 by Cancer Cells over HealthyCells

Materials and Methods:

Human cancer cell lines were purchased from the American Type CultureCollection (ATCC). They included the following: Caki-2 (renal; clearcell carcinoma), HCT-116 (colorectal carcinoma); MES-SA/Dx5 (uterinesarcoma) [all maintained in McCoy's 5a medium supplemented with 10%fetal bovine serum (FBS)], Ovcar-3 (ovarian adenocarcinoma) [maintainedin RPMI medium supplemented with 20% FBS], U87-MG (glioma) [maintainedin minimum essential medium supplemented with 10% FBS], Mia Paca-2(pancreatic carcinoma) (maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% FBS), PC-3 (prostate carcinoma) (maintained inF-12K medium supplemented with 10% FBS), MDA-MB-231 (triple-negativemammary gland adenocarcinoma) (maintained in Leibovitz's mediumsupplemented with 10% FBS), and A549 (non-small cell lung carcinoma)(maintained in F-12 medium supplemented with 10% FBS). Normal human skinfibroblasts were purchased from ATCC and grown in Fibroblast BasalMedium PCS-201-030 supplemented with serum-free kit (Fibroblast GrowthKit-Serum-Free PCS-201-040). All media (except for MDA-MB-231 cell line)also contained penicillin (100 U/ml) and streptomycin (100 μg/ml) andwere maintained at 37° C. with 5% CO₂ in air.

All cells were maintained at 37° C. in appropriate medium supplementedwith 10% FBS and 5% CO₂. Before imaging, the cells were removed fromflasks with 0.25% trypsin and were allowed to grow overnight on themicroslides VI (Ibidi). The next day, the cells were washed withphosphate-buffered saline (PBS) and were incubated with either 5 or 7.5μM (as indicated) of CLR1501 in appropriate serum-free medium for 24hours. CLR1501 is a fluorescently labeled CLR1404 analog. CLR1501 wasformulated with 0.4% of Polysorbate 20, 2% of ethanol, and saline. Afterwashing thoroughly with PBS, the cells were imaged using Bio-RadRadiance 2100 MP Rainbow laser scanning/multiphoton confocal microscopeusing a 1-s exposure time. Alternatively, cells were visualized using aNikon MR confocal microscope (Keck Laboratory, University ofWisconsin-Madison). The emission signal of CLR1501 was detected usingAlexa Fluor 488 filters (ex/em 480/520 nm).

Results:

CLR1501 was administered to five different cancer cell lines (renal,ovarian, pancreatic, melanoma, and prostate) and a normal human skinfibroblast line in vitro. Twenty-four hours later, CLR1501 exhibitedfrom five to nine-fold preferential uptake in these cancer cell lines invitro compared to normal fibroblasts (FIG. 2). Retained CLR1501 wasassociated with plasma and organelle membranes.

Example 4 Rat Glioma Model

Materials and Methods: All animals were housed and handled in accordancewith the University of Wisconsin Research Animal Resources Centerguidelines. Rat C6 glioma cells were propagated in DMEM medium (LifeTechnologies, Gaithersburg, Md.) supplemented with 10% heat-inactivatedFBS (BioWhittaker, Walkersville, Md.), 100 U/ml penicillin G, 100 mg/mlstreptomycin, and 0.01 M HEPES (Life Technologies, Gaithersburg, Md.).Intracranial tumor implantation was performed as described previously.Cohen J D, et al., Intracranial C6 glioma model in adult Wistar-Furthrats. J Neuro Oncol 1990 8(1):95-6. Briefly, 1×10⁶ C6 cells wereresuspended in 5 ml 1.2% methylcellulose and injected into the frontallobes of anesthetized female Wistar rats (Harlan, Indianapolis, Ind.).Sham-operated animals received intracranial injections of an equalvolume of methylcellulose without tumor cells.

Imaging Studies: Ten days after implantation, the presence ofintracranial tumors was confirmed with MRI. Briefly, anesthetized rats(6) received 2 ml of Gadodiamide (Gd, Omniscan 287 mg/ml, Nycomed,Princeton, N.J.) intraperitoneally and imaged 10 min later using a 1.5Tesla clinical MR system (GE Signa LX) and a GE phased array extremitycoil. The T1-weighted (TR=500 ms, TE=16.5 ms) multislice sequencescovering the entire brain of each rat were inspected to selecttumor-bearing rats with varying tumor sizes, and sham-operated rats forNM404 injections.

NM404 [18-(4-iodophenyl)-octadecylphosphocholine] (100 mg) wasradioiodinated with ¹²⁵I via isotope exchange with Na¹²⁵O in a melt ofpivalic acid. Weichert, et al. Int J Appl Rad Isotopes. 1986;37:907-913. NM404 has the same chemical structure as CLR1404 except thatit is radioiodinated with ¹²⁵I instead of ¹²⁴I or ¹³¹I. Following HPLCpurification NM404 was dissolved in an aqueous 2% Polysorbate 20solution prior to tail vein injection (5-20 μCi/200 g rat) into fourtumor-bearing and three sham-operated rats. At 1 (n=1), 2 (n=1), and 4(n=2) days after NM404 injection, animals were euthanized (CO₂) andbrains were excised and imaged on a modified Bioscan AR2000 radio-TLCscanner (1 mm increments at 2 min acquisition/lane and 1 mmhigh-resolution collimator). In addition, normal brain, blood, kidney,liver, spleen, thyroid, and tumor tissues were weighed, andradioactivity counted in a gamma counter. The tissue distribution ofradioactivity was then correlated to brain histology.

Results and Discussion: Initial imaging results with NM404 indicatedstriking uptake and prolonged retention in all gliomas ranging from 3-5mm in diameter. Radioactivity in normal brain tissue was minimal in shamoperated control animals (FIGS. 4A and 4B), whereas NM404 concentratedintensely in gliomas (FIG. 4A′-D′). Tumor to brain ratios (% injecteddose/g) in C6 glioma-bearing rats were 10.5, 12.2, and 6.7 at 24, 48,and 96 h, respectively. As has been observed in previous cell cultureand in vivo animal model studies, NM404 is apparently metabolized andeliminated from normal cells but becomes metabolically trapped in tumorcell membranes. Previous autoradiography experiments in other tumormodels have suggested that only viable tumor cells, and not normaltissue or necrotic tissues, are capable of accumulating NM404.Interestingly, even small tumors measuring a few mm in diameter, werealso detected after NM404 administration. These preliminary findingssuggest that CLR1404 may also be useful for visualization of smallinvasive tumor foci.

CONCLUSION

As has been the case in all tumor models examined previously, NM404displayed selective and prolonged retention by rat C6-gliomas evaluatedin this study.

Example 5 ¹²⁴I-CLR1404 Uptake in Various Malignant Tumors

Materials and Methods:

All described animal studies were performed according to animalprotocols approved by the Institutional Animal Care and Use Committee.Female athymic nude mice (Hsd:Athymic Nude-Foxnlnu or Crl:NU-Foxnlnu,Charles River Laboratories) about 4 to 5 weeks of age, 16 to 18 g (n=6),were used for human tumor xenograft studies. Mice were anesthetized withisoflurane and injected subcutaneously with viable tumor cells in 100 μlof Dulbecco's PBS (or, for glioma cells, 50 ml of PBS) into the rightflank. Inoculum sizes were 1×10⁶ (for renal, ovarian, glioma,pancreatic, prostate, and NSCLC models), 2=0⁶ (for colorectal anduterine models), or 3×10⁶ (breast).

Results:

Radioiodinated ¹²⁴I-CLR1404 was tested in subcutaneous and orthotopicxenografts of 60 different spontaneous, transgenic, human, and rodentmalignant cell lines and tumor types. After intravenous administration,¹²⁴ I-CLR1404 localized in almost all primary and metastatic malignanttumors regardless of anatomic location. Representative examples are ofboth human (FIG. 5A-5I) and rodent (FIG. 5J-M) tumors.

TABLE 1 Uptake of ¹²⁴I-CLR1404 in a Broad Range of Cancer Types Tumormodel Species Category Uptake* 1 Prostate PC-3 SCID mouse AdenocarcinomaYes 2 Lung A-549 (NSCLC) SCID mouse Adenocarcinoma Yes 3 Lung NCI H-69(Oat Cell) SCID mouse Small cell carcinoma Yes 4 Adrenal H-295 SCIDmouse Adenocarcinoma Yes 5 Adrenal RL-251 SCID mouse Adenocarcinoma Yes6 Colon-51 SCID mouse Adenocarcinoma Yes 7 Colon LS180 SCID mouseAdenocarcinoma Yes 8 Colon DLD1 SCID mouse Adenocarcinoma Yes 9 ColonHT-29 SCID mouse Adenocarcinoma Yes 10 Colon LS-180 Nude mouseAdenocarcinoma Yes 11 Glioblastoma U87 Nude mouse and NOD- Glioma YesSCID 12 Melanoma A-375 Nude mouse Adenocarcinoma Yes 13 Multiple myelomaNude mouse Myeloma Yes MM.1S 14 Neuroblastoma SK-N-AS Nude mouseNeuroblastoma Yes 15 Neuroblastoma NB1691 Nude mouse Neuroblastoma Yes16 Neuroblastoma CHLA-20 Nude mouse Neuroblastoma Yes 17 NeuroblastomaLan5 Nude mouse Neuroblastoma Yes 18 Ovarian HTB-77 Nude mouseAdenocarcinoma Yes 19 Ovarian Ovcar-3 Nude mouse Adenocarcinoma Yes 20Pancreatic BXPC3 Nude mouse Adenocarcinoma Yes 21 Pancreatic Mia Paca-2Nude mouse Carcinoma Yes 22 Pancreatic Capan-1 Nude mouse AdenocarcinomaYes 23 Renal cell Caki-2 Nude mouse (orthotopic) Clear cell carcinomaYes 24 Renal cell ACHN Nude mouse (orthotopic) Adenocarcinoma Yes 25Sarcoma (Meth-A) Nude mouse Fibrosarcoma Yes 26 Head and neck SCC1 Nudemouse Squamous cell Yes carcinoma 27 Head and neck SCC6 Nude mouseSquamous cell Yes carcinoma 28 Prostate LNCap Mouse Adenocarcinoma Yes29 Prostate LuCap Mouse Adenocarcinoma Yes 30 Breast MCF-7 RatAdenocarcinoma Yes 31 Triple negative breast Nude mouse AdenocarcinomaYes MDA-MB231 32 Uterine MES SA/Dx5 Nude mouse Sarcoma Yes 33Glioblastoma 22 GSC NOD-SCID mouse Glioma Yes (orthotopic) 34Glioblastoma 105 GSC NOD-SCID mouse Glioma Yes (orthotopic) 35 Breast4T1 Endogenous mouse Adenocarcinoma Yes (orthotopic) 36 Bladder SV40Mouse (orthotopic) Adenocarcinoma Yes 37 Prostate MatLyLu RatAdenocarcinoma Yes 38 Walker256 Rat Carcinosarcoma Yes 39 TRAMP prostateEndogenous mouse Adenocarcinoma Yes 40 Colon CT26 SCID mouseAdenocarcinoma Yes 41 Colon Pirc Autochthonous Pirc rat AdenocarcinomaYes 42 Min mouse intestinal Endogenous mouse Adenocarcinoma Yes 43Melanoma Mouse Adenocarcinoma Yes 44 Mammary SCC ApcMin/+ mouse Squamouscell Yes carcinoma 45 Mammary AC ApcMin/+ mouse Adenocarcinoma Yes 46Hepatocellular carcinoma Endogenous mouse Adenocarcinoma Yes 47 GliomaL9 Rat xenograft Glioma Yes 48 Glioma C6 Rat xenograft Glioma Yes 49Glioma CNS1 Rat xenograft Glioma Yes 50 Glioma RG2 Rat xenograft GliomaYes 51 Retinoblastoma Endogenous mouse Blastoma Yes 52 Pancreatic c-mycEndogenous mouse Adenocarcinoma Yes 53 Pancreatic Kras Endogenous mouseAdenocarcinoma Yes 54 Cervical-HPV Endogenous mouse Adenocarcinoma Yes55 Esophageal Endogenous Mouse Adenocarcinoma Yes 56 Intestinal polypEndogenous mouse Adenoma (benign) No 57 Mammary alveolar Endogenousmouse Hyperplasia (benign) No hyperplasia 58 Hepatoma Hep-3B Nude mouseCarcinoma No 59 Hepatoma Hep-G2 Nude mouse Carcinoma No 60 Pirc ratcolon adenoma Pirc rat Adenoma No *Tumor uptake was considered positiveif tumor to muscle ratio was greater than 3. Tumor:muscle ratio lessthan or equal to 2 was considered negative.

Example 6 Clinical Trial Evaluating Patients with Non-Small Cell LungCarcinoma (“NSCLC”) using CLR1404

Although CLR1404 has displayed selective and prolonged tumor retentionin 55/60 xenograft and spontaneous rodent models, a physician sponsoredIND initiated clinical evaluation of the agent in Stage 4 human NSCLCpatients in order to determine whether or not it would exhibit similartumor uptake and retention properties in humans. To date, two patientswith advanced NSCLC were imaged after an injection of <1 mCi of¹³¹I-CLR1404. Blood and urine samples were collected at predeterminedtimes, and gamma imaging performed at several time points followingadministration. In both patients, significant tumor uptake and retentionof CLR1404 was demonstrated in the primary lung tumor, as seen in FIG.6. Relative to the high liver uptake values seen previously with itsfirst generation predecessor, NM324, liver and abdominal activity aremuch lower with CLR1404, suggesting the feasibility of evaluating thisagent in other abdominal cancers including pancreatic, colon, andprostate.

Materials and Methods: Following intravenous injection of iodine-131labeled CLR1404 (1 mCi/20 μg), patients with advanced NSCLC wherescanned at 3, 6, 24, 48, 96 h and at 7 and 11 days on a GE Maxxus dualHead SPECT scanner. Blood and urine samples were collected forpharmacokinetic analysis as well as clinical hematologic, renal, andhepatic bioanalysis.

Results: Initial qualitative imaging results indicate that iodine-131labeled CLR1404 clearly localizes in bilateral pulmonary masses as earlyas 24 h after injection and is selectively retained in these tumors inexcess of 11 days. Moreover, background radioactivity in the liver andlower abdominal region including urinary bladder, kidneys, andintestines was significantly less than was observed previously with itspredecessor, NM324. No adverse reactions were observed in any of thepatients.

Conclusions: These preliminary findings suggest that CLR1404 exhibitssimilar tumor uptake and retention properties in human NSCLC as was seenpreviously in rodent models. Although based on only two patients at thispoint, it appears that CLR1404 does indeed localize in and undergoselective and prolonged tumor retention in human non-small cell lungcancer.

Patient 1: 55 year old male with bilateral 3 cm left lobe andinfiltrative right lobe NSCLC and a brain metastasis and a small rightadrenal mass. He has participated in numerous standard and experimentaltreatment regimens. Images are shown in FIG. 6A-C.

Patient 2: 70 year old male recently diagnosed with 6 cm upper lobenon-small cell lung carcinoma, a 5 cm liver mass, an iliac bonemetastasis and a very small brain metastasis. He had recently completedlow dose carboplatin/taxol chemotherapy and palliative radiotherapy tothe iliac and brain metastases the week prior to initiating the CLR1404trial. Images are shown in FIG. 6D-G.

Example 7 Detection of 3 Previously Unknown Brain Tumor Metastases inNSCLC Patient Using ¹²⁴I-CLR1404

Materials and Methods:

Human PET brain scans were acquired on a 64-slice PET/CT scanner(Discovery VCT, General Electric) at multiple time points after theinjection of about 5 mCi of ¹²⁴I-CLR1404 using a 90-min dynamicacquisition sequence (2D, nine frames at 10 min each, VIP list mode on)and reconstructed [Advantage Workstation version AW4.4, GeneralElectric, 30 cm DFOV (display field of view), 128×128, OSEM VUE Point,10 subsets with two iterations, standard z axis, attenuation correctionand dead time, scatter, and decay correction].

Results:

Preliminary results were obtained in an NSCLC patient withoutneurological symptoms using ¹²⁴I-CLR1404 PET/CT. Imaging revealed threepreviously unknown brain lesions highly suspicious for metastases thatwere subsequently confirmed with gadolinium-enhanced MRI (FIG. 7).

Example 8 Detection of Tumor Recurrence of a Right Frontal FalcineMetastasis Using ¹²⁴I-CLR1404

Recurrent brain metastasis in 60-year-old woman with malignant melanoma.Magnetic resonance (“MR”) (FIG. 8A) and ¹²⁴I-CLR1404 PET images (FIG.8B) and images 8 months after stereotactic radiosurgery for tumorrecurrence of a right frontal falcine metastasis (FIG. 8C) shows a focusof abnormal activity with CLR1404 (arrow). Corresponding enhancing focuson initial MR imaging was interpreted as radiation necrosis versuspossible recurrence. Subsequent MR imaging showed further increase insize of the nonspecific enhancing lesion, coupled with increasedperilesional edema indicating a recurrence of the malignant tumor. Theseresults indicate that ¹²⁴I-CLR1404 was sequestered by cancer cells thatwere resistant to the radiosurgery and eventually established arecurrent tumor.

Compounds of the present invention include anti-cancer drugs linked tothe CLR1404 core molecule. These compounds are capable of targetingcancer cells and cancer stem cells including brain cancer cells suchthat the anti-cancer drug is sequestered and retained by the cancercell. These compounds provide the first targeted treatment of cancercapable of being adapted to specifically administer a range ofanti-cancer drugs to cancer cells to both treat the cancer and preventmetastasis and recurrence.

Example 9 Paclitaxel-Conjugates and IC50 for Various Cancer Cell Lines

Method

Cancer cell lines including, MDA-MB-468 (Breast), NCI-H1299 (Lung),NCI-H460 (Lung), Capan-2 (Pancreas), MiaPaCa-1 (Pancreas), HT29(Colorectal), HCT116 (Colorectal) and PC-3 (Prostate) were treated withserial concentrations of paclitaxel and CLR1404-paclitaxel conjugates(i.e. CLR1601, CLR1602 and CLR1603). The cell lines were then measuredfor cell viability and reported as IC50 for each treatment.

Results

CLR1601 and CLR1603 were capable of reducing cell viability for each ofMDA-MB-468 (Breast), NCI-HI299 (Lung), NCI-H460 (Lung) Capan-2(Pancreas), MiaPaCa-1 (Pancreas), HT29 (Colorectal), HCT116 (Colorectal)and PC-3 (Prostate) cancer cell lines. See FIGS. 9-16, respectively.IC50 for each paclitaxel-1404 conjugate (i.e. CLR1601 and CLR1603) andpaclitaxel are reported in Table 2. IC50 for CLR1602 is not shown,however CLR1602 was not capable of significantly reducing cancer cellline viability because CLR1602 is non-hydrolyzable. In vivo, freepaclitaxel is taken up by cancerous tumor cells at a much lower rate dueto the non-specific nature of paclitaxel uptake. Thus, in vivo, theamount of PLE-paclitaxel conjugate necessary for cancer cell deathshould be on par or less than that for paclitaxel and may result in agreatly reduced toxicity to non-cancer cells.

TABLE 2 IC50 for CLR1601, CLR1603 and Paclitaxel CLR1601 CLR1603Paclitaxel MDA-MB-468 (Breast) 3.77 nM 3.42 nM  1.9 nM NCI-HI1299 (Lung)60.3 nM  108 nM 1.82 nM NCI-H460 (Lung) 29.5 nM 171.1 nM  1.66 nMCapan-2 (Pancreas) 56.7 nM 83.6 nM 7.91 nM MiaPaCa-1 (Pancreas) 37.3 nM38.9 nM 1.32 nM HT29 (Colorectal) 92.2 nM 70.6 nM 1.07 nM HCT116(Colorectal)  7.6 nM   11 nM 0.87 nM PC-3 (Prostate) 34.4 nM 29.5 nM 0.9 nM

Example 10 Flow Cytometry Assays

Methods

Annexin V and PI (phosphatidylinositide) staining by flow cytometry wasutilized to determine percentages of live, early apoptotic, lateapoptotic, and necrotic cells. In short, cells were treated withcytotoxic agents and stained with an Annexin V/PI labeling kit (LifeTechnologies). Cells were analyzed on an LSRII flow cytometer (BDBiosciences). As shown in Tables 3-9, cells were classified as: Live(Annexin V negative, PI negative), Early apoptotic (Annexin V positive,PI negative), Late apoptotic (Annexin V positive, PI positive), andNecrotic (Annexin V negative, PI positive). A representative scatterplot is shown in FIG. 17 for MDA-MB-468 cells treated with 5 μM ofCLR1601 for 72 hours. Annexin V (attached to AlexaFluor 488) is shown onthe x-axis, while PI is shown on the y-axis. The lower left quadrantindicates live cells, the upper left quadrant indicates necrotic cells,the upper right quadrant indicates late apoptotic cells and the lowerright quadrant indicates early apoptotic cells. Debris was eliminatedfrom this analysis.

Results

MDA-MB-468 cells, a triple negative breast cancer cell line, weretreated with CLR conjugates (CLR1601 and CLR1603) for 72 hours and withpaclitaxel (“PTX”) for 24 hours. See Table 3. MDA-MB-468 cells, werealso treated with CLR conjugates (CLR1606 and CLR1607) for 72 hours andwith geldanamycin (“GEL”) for 48 hours. See Table 4. For PTX conjugates,cell viability was reduced from 61.1% (no drug treatment) to 17.0%,17.8%, 22.2%, 19.7%, 53.8%, and 48.9% after treatment with 1 μM CLR1601,5 μM CLR1601, 1 μM CLR1603, 5 μM CLR1603, 100 nM PTX, and 1 μM PTX,respectively. See Table 3. For GEL conjugates, cell viability wasreduced from 61.1% to 58.8%, 42.7%, 52.7%, 56.9%, and 26.2% aftertreatment with 1 μM CLR1606, 10 μM CLR1606, 1 μM CLR1607, 10 μM CLR1607,and 1 μM GEL, respectively. See Table 4.

TABLE 3 MDA-MB-468 Treated with Paclitaxel Conjugates for 72 HoursMDA-MB-468 72 hrs CLR1601 CLR1601 CLR1603 CLR1603 PTX PTX (% of totalcells) No Drug (1 uM) (5 uM) (1 uM) (5 uM) (100 nM) (1 uM) Live 61.1 1717.8 22.2 19.7 53.8 48.9 Necrotic 2.83 15.8 21.6 22 22.5 8.89 10.8 LateApoptotic 20.5 57.5 57.9 49.1 55.2 31.4 34.8 Early Apoptotic 15.5 9.832.71 6.74 2.65 5.9 5.5

TABLE 4 MDA-MB-468 Cells Treated with Geldanamycin Conjugates for 72Hours MDA-MB-468 72 hrs CLR1606 CLR1606 CLR1607 CLR1607 GEL (% of totalcells) No Drug (1 uM) (10 uM) (1 uM) (10 uM) (1 uM) Live 61.1 58.8 42.752.7 56.9 26.2 Necrotic 2.83 2.31 19.4 1.62 1.63 16.3 Late Apoptotic20.5 25.7 33.8 25.1 26.4 37.9 Early Apoptotic 15.5 13.2 4.13 20.6 15.119.6

COLO 829 cells, a melanoma cell line, were treated with GEL conjugates(CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. See Table 5. Cellviability was reduced from 80.8% (no drug treatment) to 70.4%, 21.1%,67.9%, 54.3%, 32.4%, and 18.6% after treatment with 1 μM CLR1606, 10 μMCLR1606, 1 μM CLR1607, 10 μM CLR1607, 100 nM GEL, and 1 μM GEL,respectively. See Table 5.

TABLE 5 COLO 829 Cells Treated with Geldanamycin Conjugates for 72 HoursCOLO 829 72 hrs CLR1606 CLR1606 CLR1607 CLR1607 GEL GEL (% of totalcells) No Drug (1 uM) (10 uM) (1 uM) (10 uM) (100 nM) (1 uM) Live 80.870.4 21.1 67.9 54.3 32.4 18.6 Necrotic 1.74 1.59 28.6 2.36 16.1 4.9 6.42Late Apoptotic 4.18 6.7 40.1 9.05 20.9 40.8 53.7 Early Apoptotic 13.321.3 10.2 20.7 8.69 21.9 21.4

PANC-1 cells, a pancreatic cancer cell line, were treated with GELconjugates (CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. SeeTable 6. Cell viability was reduced from 44.2% (no drug treatment) to42.0%, 21.5%, 44.0%, 33.0%, 23.3%, and 18.9% after treatment with 1_(μM CLR)1606, 10 μM CLR1606, 1 μM CLR1607, 10 μM CLR1607, 100 nM GEL,and 1 μM GEL, respectively. See Table 6.

TABLE 6 PANC-1 Cells Treated with Geldanamycin Conjugates for 72 HoursPANC-1 72 hrs CLR1606 CLR1606 CLR1607 CLR1607 GEL GEL (% of total cells)No Drug (1 uM) (10 uM) (1 uM) (10 uM) (100 nM) (1 uM) Live 44.2 42 21.544 33 23.3 18.9 Necrotic 47.9 46.8 65.3 48.1 54.7 59.4 66.4 LateApoptotic 5.65 8.63 12.6 4.83 9.81 12.6 12.8 Early Apoptotic 2.23 2.590.63 3.1 2.43 4.68 1.93

22RV1 cells, a prostate cancer cell line, were treated with GELconjugates (CLR1606 and CLR1607) for 72 hrs and GEL for 48 hours. SeeTable 7. Cell viability was 20.3%, 21.3%, 16.0%, 21.9%, 15.7%, 19.4%,and 28.1% after treatment with no drug, 1 μM CLR1606, 10 μM CLR1606, 1μM CLR1607, 10 μM CLR1607, 100 nM GEL, and 1 μM GEL, respectively. SeeTable 7. Basal cell death was high with this cell line; the cells didnot respond well to the method of cell collection.

TABLE 7 22RV1 Cells Treated with Geldanamycin for 72 Hours 22RV1 72 hrsCLR1606 CLR1606 CLR1607 CLR1607 GEL GEL (% of total cells) No Drug (1uM) (10 uM) (1 uM) (10 uM) (100 nM) (1 uM) Live 20.3 21.3 16 21.9 15.719.4 28.1 Necrotic 49.5 51.7 57.7 55.2 59.1 47.1 46.6 Late Apoptotic26.2 23.4 22.9 20.7 23.1 28.4 19.1 Early Apoptotic 4.1 3.63 3.44 2.272.19 5.09 6.22

CLR conjugates appear to require a longer treatment period with cells toinduce cell death. Treatment of MDA-MB-468 cells with 1 μM CLR1601 and 1μM CLR1603 for 48 hours resulted in a reduction of cell viability from86.2% (no drug treatment) to 79.4% and 81.4%, respectively. See Table 8.Treatment of COLO 829 cells with 1 μM CLR1606 and 1 μM CLR1607 for 48hours resulted in a reduction of cell viability from 91.7% (no drugtreatment) to 90.1% and 82.7%, respectively. See Table 9.

TABLE 8 MDA-MB-468 Treated with Paclitaxel Conjugates for 48 HoursMDA-MB-468 (% of 48 hrs CLR1601 CLR1603 PTX PTX total cells) No Drug (1uM) (1 uM) (100 nM) (1 uM) Live 86.2 79.4 81.4 77.9 73.3 Necrotic 2.8713.1 12.4 14.1 15.6 Late Apoptotic 8.33 6 4.78 5.54 9.18 Early 2.56 1.491.4 2.47 1.97 Apoptotic

TABLE 9 COLO 829 Cells Treated with Geldanamycin Conjugates for 48 HoursCOLO 829 (% of 48 hrs CLR1606 CLR1607 GEL GEL total cells) No Drug (1uM) (1 uM) (100 nM) (1 uM) Live 91.7 90.1 82.7 36.1 24.6 Necrotic 5.966.91 8.94 22.7 22.7 Late Apoptotic 1.67 2.2 5.54 29 38.3 Early 0.72 0.792.77 12.2 14.4 Apoptotic

Overall, PLE-paclitaxel and PLE-geldanamycin conjugates were shown to becapable of reducing tumor cell viability including inducing cell deathfor a variety of tumor types.

What is claimed is:
 1. A method of treating breast cancer, lung cancer,pancreatic cancer, colorectal cancer, and/or prostate cancer comprisingadministering an effective amount of a therapeutic compound of Formula(V),

to a subject with cancer.
 2. The method of claim 1 wherein the cancercomprises cancer stem cells.
 3. The method of claim 1 wherein the canceris recurrent.